Factor V Leiden Heterozygotes Research Articles

Practice patterns of the medical evaluation of living liver donors in the United States.

Living donor liver transplantation (LDLT) can help address the growing organ shortage in the United States, yet little is known about the current practice patterns in the medical evaluation of living liver donors. We conducted a 131-question survey of all 53 active LDLT transplant programs in the United States to assess current LDLT practices. The response rate was 100%. Donor acceptance rate was 0.33 with an interquartile range of 0.33-0.54 across all centers. Areas of high intercenter agreement included minimum age cutoff of 18 years (73.6%) and the exclusion of those with greater than Class 1 obesity (body mass index, 30.0-34.9 m/kg 2 ) (88.4%). Diabetes mellitus was not an absolute exclusion at most centers (61.5%). Selective liver biopsies were performed for steatosis or iron overload on imaging (67.9% and 62.3%, respectively) or for elevated liver enzymes (60.4%). Steatohepatitis is considered an exclusion at most centers (84.9%). The most common hypercoagulable tests performed were factor V Leiden (FVL) (88.5%), protein C (73.1%), protein S (71.2%), antithrombin III (71.2%) and prothrombin gene mutation (65.4%). At 41.5% of centers, donors were allowed to proceed with donation with FVL heterozygote status. Most programs discontinue oral contraceptive pills at least 28 days prior to surgery. At most centers, the need for cardiovascular ischemic risk testing is based on age (73.6%) and the presence of one or more cardiac risk factors (68.0%). Defining areas of practice consensus and variation underscores the need for data generation to develop evidence-based guidance for the evaluation and risk assessment of living liver donors.

Safety of DIEP Flap Reconstruction in Patients with Factor V Leiden: A Retrospective Cohort Study.

Background:Factor V Leiden (FVL) is the most common inherited thrombophilia in White people. Thrombotic complications resulting from free flap breast reconstruction in FVL patients have been studied to a limited degree. We evaluated whether patients heterozygous for a FVL mutation undergoing deep inferior epigastric perforator flap reconstruction had increased risk of micro- or macrovascular thrombotic complications compared with patients without a diagnosed thrombophilia.Methods:We performed a retrospective cohort study of deep inferior epigastric perforator flap reconstructions at Brigham and Women’s Hospital (1/2015–12/2020) comparing patients diagnosed as FVL heterozygotes compared with matched controls without a diagnosed thrombophilia. Patients were matched using coarsened exact matching algorithm based on clinical characteristics. The primary outcomes were micro- (return to OR for flap compromise, flap loss) and macrovascular (venous thromboembolism) complications.Results:A total of 506 patients (812 flaps) were included in this study. Eleven patients (17 flaps) were FVL heterozygotes. After matching, 10 patients (16 flaps) with FVL were matched to 55 patients (94 flaps). The return to OR for flap compromise was 0% in the FVL cohort compared with 5% (n = 5/94, 3/94 flaps lost, P = 1.00) in the matched controls (1.9%, n = 15/795 in unmatched controls, 0.6%, n = 5/795 loss rate). There were zero venous thromboembolism events among FVL patients compared with 2% of controls (n = 1/55).Conclusions:FVL heterozygosity did not increase the risk of micro- or macrovascular complications in patients undergoing deep inferior epigastric perforator flap breast reconstruction. This study supports the safety of microvascular reconstruction in this group of patients when appropriate venous thromboembolism prophylaxis is given.

Gene polymorphism of blood coagulation factors and endothelial dysfunction in early and late preeclampsia.

The aim of our study was to find out the influence of gene polymorphisms of coagulation factors, endothelial dysfunction and regulators of blood pressure in early (EPE) and late preeclampsia (LPE). The study of genetic polymorphisms of blood coagulation factors and fibrinolysis (1691 G/A factor V Leiden (FVL), 20210 G/A prothrombin, -675 5G/4G PAI-1, 455 G/A fibrinogen β), endothelial dysfunction (192 Q → R paraoxonase 1, 677 C/T MTHFR, arterial pressure regulator (235 M/T angio­tensinogen II (AGT II)) using an PCR was performed. A prospective cohort study of 39 women with EPE, 93 with LPE and 44 pregnant women with a physiological pregnancy (C group) was conducted. The average gestational age at the time of delivery in EPE group was lower than in LPE and control group (p<0.001). In group with EPE new-borns had low weight-growth characteristics, low grade by the Apgar scale and foetal distress (38.5% vs. 9.7%, p<0.05). Caesarean section in EPE group was performed by 2.25 times more often than in control group and by 2,13 times than in LPE group (p<0,05). It was detected that the number of 1691 GA FVL heterozygote carriers in the group with EPE was significantly higher than in LPE group (p<0.05, OR=3.65, 95% CI 1.5-8.9) and control group (6.04, 1.7-21.6). The number of 20210 GG homozygotes and 20210 GA heterozygotes in prothrombin gene was probably lower in EPE group compared with the LPE and control group (0.03, 0.002-0.49, and 0.18, 0.06-0.53, respectively). It was established increase in frequency of 677 TT MTHFR genotype in EPE compared with control group (17.27; 0.9-317). Also, the carriers of 235T allele AGT II gene have an increased risk of EPE and PPE development (2.25, 1.2-4.2), (1.9, 1.1-3.3) respectively. The allele -455A of fibrinogen β gene increases the chances of developing EPE by 4.4 times (2.0-9.5), and LPE by 3.5 times (1.7-7.1). Risk factors that significantly increase the chances of developing early preeclampsia were identified: allele 1691 A of FV Leiden (5.96, 1.5-8.9), allele 20210 A of prothrombin (39.8, 2,3-679), 677T MTHFR (2.5, 1.18-5.3). In was detected that other researched polymorphisms between groups with PE were not significantly different and did not affect on time of preeclampsia development.

ASSOCIATION BETWEEN FACTOR V LEIDEN (FV G1691A) MUTATION AND CORONARY ARTERY DISEASE DEVELOPMENT IN GAZA STRIP - PALESTINE

Introduction: Coronary Artery Disease(CAD) is multi-factorial and usually results from a combination of acquired environmental and inherited factors with a change in lifestyle. One of these inherited factors is the G1691A factor V. Factor V Leiden (FVL) is the most common heritable thrombophilic disorder. It is concomitant with an increased the risk of thrombosis and may lead to CAD. The aim of this study is to investigate the relation of this mutation with CAD in Gaza strip population. Methods: it is a retrospective study which includes 180 samples; patients with CAD, n=90 and control group, n=90. An interview with questionnaire was applied. EDTA samples were collected for DNA extraction. The FVL mutation was identified by PCR-RFLP. Results: The frequency of FVL genotypes were: wild type 83.2%, heterozygous16.7% and complete absence of mutant homozygous in control group, whereas in CAD patients were: 70%,26.7% and 3.3%. The FVL G and A alleles frequencies of the participants were: for case group 0.833 and 0.167 while 0.917 and 0.083 were in control group. Furthermore, the distribution of FVL heterozygote and mutant(AA) genotypes were not significantly different between the study and control groups. The GA and AA genotypes increase the risk of developing CAD in patients by 1.9 and 8.3 times respectively. The presence of FVL A allele increases the risk of exposure to heart attack among CAD patient (OR =1.7). The frequency of A allele among diabetic patients increases the risk of developing CAD 1.5 fold. Conclusion: Factor V Leiden A allele is associated with the development CAD.

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα.

Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

Abstract 35: Identification of a Mouse Strain Modifier Locus for Factor V Leiden/Tissue Factor Pathway Inhibitor Dependent Thrombosis

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p<0.001) which suggested that multiple modifier genes were contributing to the phenotype. To isolate individual dominant modifier loci, we adopted a serial backcrossing strategy in which F1 FVQ/Q Tfpi+/- mice were serially backcrossed to B6 FVQ/Q mice for 17 generations. A standard mouse positional cloning strategy was used on the 110 surviving FVQ/Q Tfpi+/- mice from these crosses which identified a chromosome 2 modifier locus (148-169 Mb) under genetic selection. Currently there are 16 genes in this locus with known annotated non-synonymous coding variants. Ongoing studies are focused on identifying the responsible genetic DBA/2J variant within this locus. Identification of this modifier gene will represent a significant advance for the genetics of thrombotic disease. Not only will it give insight into the pathways leading to thrombosis, it may also reveal previously unexplored therapeutic targets for the prevention of thrombotic events.

Influence of single nucleotide polymorphisms on thrombin generation in factor V Leiden heterozygotes

Carriership of the factor V (FV) Leiden mutation increases the risk of venous thromboembolism (VTE) ~4-fold, but the individual risk of each FV Leiden carrier depends on several co-inherited risk and protective factors. Under the hypothesis that thrombin generation might serve as an intermediate phenotype to identify genetic modulators of VTE risk, we enrolled 188 FV Leiden heterozygotes (11 with VTE) and determined the following parameters: thrombin generation in the absence and presence of activated protein C (APC); plasma levels of prothrombin, factor X, antithrombin, protein S and tissue factor pathway inhibitor; and the genotypes of 24 SNPs located in the genes encoding these coagulation factors and inhibitors. Multiple regression analysis was subsequently applied to identify the (genetic) determinants of thrombin generation. The endogenous thrombin potential (ETP) showed a striking inter-individual variability among different FV Leiden carriers and, especially when measured in the presence of APC, correlated with VTE risk. Several SNPs in the F2 (rs1799963, rs3136516), F10 (rs693335), SERPINC1 (rs2227589), PROS1 (Heerlen polymorphism) and TFPI (rs5940) genes significantly affected the ETP-APC and/or the ETP+APC in FV Leiden carriers. Most of these SNPs have shown an association with VTE risk in conventional epidemiological studies, suggesting that the genetic dissection of thrombin generation leads to the detection of clinically relevant SNPs. In conclusion, we have identified several SNPs that modulate thrombin generation in FV Leiden heterozygotes. These SNPs may help explain the large variability in VTE risk observed among different FV Leiden carriers.

Deep Vein Thrombosis, Ecythyma Gangrenosum and Heparin-Induced Thrombocytopenia Occurring in a Man with a Heterozygous Factor V Leiden Mutation

Skin necrosis and limb gangrene are occasional thrombotic manifestations of anticoagulation therapy. We report a man heterozygous for the Factor V Leiden (FVL) mutation, and with a history of recurrent deep venous thrombosis, who initially presented with a necrotic skin lesion of the right flank while on warfarin therapy with a therapeutic international normalized ratio. Warfarin was discontinued and he received intravenous heparin. Thereafter he developed thrombocytopenia and pedal erythema and was diagnosed with heparin-induced thrombocytopenia (HIT). Heparin was replaced with argatroban. He ultimately underwent bilateral below-knee amputations for the thrombotic complications of the HIT. The initial necrotic lesion healed with antibiotics and wound care. Pathologic examination of multiple biopsy specimens revealed two separate lesions. One was necrotic tissue infiltrated with methicillin resistant Staphylococcus aureus having features of ecthyma gangrenosum. The second showed thrombotic changes consistent with HIT. The case illustrates the differential diagnosis of skin necrosis and limb gangrene in patients on warfarin and heparin, and also the clinical complexities that can occur in a FVL heterozygote.

The Role of the Factor V Leiden Mutation in Osteonecrosis of the Hip

We examined the hypothesis that the factor V Leiden (FVL) and G20101A prothrombin gene mutations are commonly associated with hip osteonecrosis. We prospectively evaluated 244 consecutively referred adults with osteonecrosis (ON), 161 idiopathic and 83 secondary. Cases (n = 244) did not differ from 104 normal controls by race. Of the 244 patients, 23 (9.4%) were FVL heterozygotes versus 2 of 104 controls (1.9%), P = .013, risk ratio (RR) = 4.90, 95% confidence interval (CI) 1.18 to 20.4. Of the 161 patients with idiopathic ON, 15 (9.3%) were FVL heterozygotes versus 2 of 104 normal controls (1.9%), P = .017, RR = 4.84, 95% CI 1.13 to 20.8. Of the 83 patients with secondary ON, 8 (9.6%) FVL heterozygotes versus 2 of 104 normal controls (1.9%), P = .024, RR = 5.01, 95% CI 1.09 to 23.0. Prothrombin gene heterozygosity in normal controls (2.9%) did not differ from ON cases (3.4%), P = 1.0. The thrombophilic FVL mutation is commonly associated with and may be pathoetiologic for hip osteonecrosis.

Variable plasma levels of Factor V Leiden correlate with circulating platelet microparticles in carriers of Factor V Leiden

Introduction Inheritance of Factor V Leiden (FVL) is associated with an increased but variable level of risk for thrombosis. We have previously shown that FVL heterozygotes have elevated levels of circulating pro-coagulant microparticles (MP). Here we sought to determine if these subjects differed in their plasma levels of FVL and if this was related to MP concentrations and/or history of thrombosis. Materials and Methods The Hemoclot Quanti. V-L clotting assay was used to specifically measure FVL in plasma samples from 44 known carriers (12 M, 32 F; aged 46 ± 13 years). Circulating MP were quantified by flow cytometry using fluorochrome conjugated antibodies to platelet (CD41a), leukocyte (CD45), and endothelial (CD62e) surface markers, and MP prothrombinase activity was determined by ELISA. Results The cohort was found to have a mean FVL of 49.5 ± 5.6% and this was positively correlated to the total number of circulating CD41a + MP (R = 0.31, p = 0.03) but not to other MP subsets or to MP prothrombinase activity. The amount of FVL relative to normal factor V (FVL/FV clotting ratio) was calculated and found to be highly variable, ranging from 0.37 to 0.69, and significantly correlated with a history of thrombosis (n = 14; p = 0.04). Conclusions This is the first study to investigate the relationship between varying levels of FVL and plasma derived MP. These results are consistent with our previous findings of an increase in MP levels in carriers of FVL as compared to controls, and suggest a role for FVL/FV ratio in predicting risk of thrombosis in carriers of FVL.

Avascular Villi, Increased Syncytial Knots, and Hypervascular Villi Are Associated with Pregnancies Complicated by Factor V Leiden Mutation

There is controversy about whether pathologic abnormalities are associated with pregnancies complicated by factor V Leiden (FVL) mutation. The purpose of this study was to evaluate 105 placentas delivered to mothers heterozygous for FVL mutation to determine if there are pathologic changes suggestive of hypoxia or thrombosis, which correlate with mutation status. We examined placentas obtained as part of a prospective study of 5188 pregnancies analyzed for the presence of FVL mutation in either the mother or the infant. One hundred five placentas from mothers heterozygous for the mutation were compared with 225 controls matched for maternal age, race, and geographic site. Of the 330 pregnancies, 50 infants were FVL mutation heterozygotes. Maternal FVL heterozygote status was associated with more frequent increased numbers of syncytial knots (13% vs 4%); the difference remained significant after controlling for hypertension, preeclampsia, small-for-gestational-age infants, and delivery prior to 35 weeks of gestation (odds ratio 3.6, 95% confidence interval 1.5-8.7, P = 0.004). Maternal FVL heterozygotes had more hypervascular villi (10% vs 3%), with significance retained controlling for delivery route (odds ratio 3.4, 95% confidence ratio 1.2-9.4, P = 0.018). Placentas from infants heterozygous for FVL mutation had more avascular villi than controls (odds ratio 2.9, 95% confidence interval 1.5-5.6, P = 0.001). Fetal or maternal FVL heterozygosity was not associated with infarcts, small-for-gestational-age placentas, or fetal thrombotic vasculopathy. This analysis demonstrates that pathologic findings associated with placental hypoxia, specifically focal avascular villi, increased numbers of syncytial knots, and hypervascular villi, also correlate with FVL heterozygosity in infants or mothers.

Antiphospholipid antibodies and pregnancy outcomes in women heterozygous for Factor V Leiden

Antiphospholipid antibodies are associated with a spectrum of pregnancy complications, including preeclampsia and small for gestational age (SGA) fetuses. We sought to assess anticardiolipin and anti-beta2-glycoprotein I (anti-beta2-GPI) IgG and IgM antibody prevalence and the relationship of these antibodies to pregnancy complications in women with the Factor V Leiden (FVL) mutation. The study comprised a secondary analysis of a multicenter, prospective observational study of FVL prevalence among 5188 asymptomatic pregnant women. A subset of 362 women (117 FVL heterozygotes, 245 matched controls) had serum collected at the time of the original study and underwent serum analysis for anticardiolipin and anti-beta2-GPI IgG and IgM as a part of this analysis. The primary outcome was preeclampsia and/or SGA (<10%). The overall prevalence of anticardiolipin and anti-beta2-GPI IgG and IgM antibodies was low and did not vary with FVL status. Forty-seven women (13.0%) developed preeclampsia and/or SGA. There were no differences in primary outcome rates between women with and without aPL antibodies, regardless of FVL mutation status. Among FVL carriers, the presence of antiphospholipid antibodies does not appear to contribute to adverse pregnancy outcome.

519: Do antiphospholipid antibodies affect pregnancy outcomes in women heterozygous for Factor V Leiden?

Antiphospholipid antibodies are associated with a spectrum of pregnancy complications, including preeclampsia (PE) and small for gestational age (SGA) fetuses. Our goals were to assess the frequency of elevated levels of anticardiolipin (aCL) and anti-2-glycoprotein I (anti-2-GPI) IgG and IgM antibodies in women with and without the Factor V Leiden (FVL) mutation and to examine if interactions between the FVL mutation and aCL and anti-2-GPI antibodies alter the rates of PE and SGA. Secondary analysis of a multicenter, prospective observational study of FVL prevalence among 5188 pregnant women. 371 women (120 cases of FVL heterozygotes and 251 FVL mutation negative randomly selected controls [matched for clinical center, maternal age, and race/ethnicity]) underwent serum analysis for the presence of aCL and anti-2-GPI IgG and IgM. The primary outcome was development of PE and/or SGA (<10%). Forty seven women (12.7%) developed the primary outcome; there were no differences in primary outcome rates between women with and without antiphospholipid antibodies, regardless of FVL mutation status. Among FVL carriers, antiphospholipid antibody status does not appear to contribute to adverse pregnancy outcome.

Predicting Thrombosis in Factor VLeiden Heterozygotes.

Factor VLeiden (G1691A;R506Q) is an autosomal dominant allele displaying high prevalence (3–7%) in the United States Caucasian population and a high incidence of venous thrombosis in homozygotes (50% lifetime risk) but reduced penetrance in heterozygote carriers (<10% lifetime risk). Factors that precipitate or suppress the expression of the thrombotic phenotype in factor VLeiden heterozygotes are not well defined except for relatively infrequent instances of coexpression of other thrombophilic risk factors, e.g. the prothrombin G20210A mutant. Our goal is to mathematically evaluate the effect of factor VLeiden on tissue factor-initiated thrombin generation and determine whether concentration variations within the normal range of antithrombin (86–128%), prothrombin (60–140%) or protein C (77–183%) modulate the factor VLeiden effect when all other factor levels are at mean physiologic concentrations. Our mathematical model (Hockin et al. (2002) JBC 277:18322) was extended to incorporate the protein C pathway by including descriptions of thrombin binding to thrombomodulin, activation of protein C and factor Va inactivation by activated protein C. Simulations were conducted with 100% factor V, 50% factor V/50% factor VLeiden (heterozygote), and 100% factor VLeiden (homozygote). Heterozygous expression of factor VLeiden increases the maximum level of thrombin by 25% and the maximum rate of thrombin generation by a factor of 1.6 over that seen with 100% factor V. Homozygous factor VLeiden yields a 2.6-fold increase in maximum rate and a 60% increase in maximum thrombin level compared to 100% factor V. Decreasing the protein C concentration to 77% in a factor VLeiden heterozygote results in a thrombin generation profile with maximum levels and rates comparable to the factor VLeiden homozygote. At 150% protein C, thrombin generation is suppressed to levels similar to those seen with 100% factor V. When the effect of variable prothrombin levels in a factor VLeiden heterozygote is examined, a 60% level of prothrombin decreases the maximum level of thrombin generation below that seen with 100% factor V. When prothrombin levels are at 140% in the factor VLeiden heterozygote, the thrombin generation profile is similar to that observed with 100% factor VLeiden . When the effect of variable antithrombin levels was tested in a factor VLeiden heterozygote, a 14% decrease in antithrombin resulted in maximum thrombin levels approaching those of the factor VLeiden homozygote. In contrast, 128% antithrombin in a factor VLeiden heterozygote resulted in the suppression of thrombin generation to levels consistent with those seen with 100% factor V. Our results predict that the thrombotic effect of heterogenous expression of factor VLeiden can be reduced when prothrombin levels are at the low extreme of the normal range or when antithrombin or protein C levels are at the high extreme of the normal range. Conversely, the thrombotic effect of the heterozygous expression of factor VLeiden is intensified when prothrombin levels are at the high extreme of normal values or when antithrombin or protein C levels at the low extreme of normal. These results may contribute to the understanding of phenotypic variation within factor VLeiden heterozygotes.

Evidence of Increased Plasma Coagulative Capacity by CloFAL Assay among Pediatric Factor V Leiden and Prothrombin G20210A Heterozygotes with, Versus without, a First-or Second-Degree Family History of Venous Thromboembolism

BACKGROUND: The factor V (FV) Leiden and prothrombin (PT) G20210A polymorphisms in heterozygous state are present in 5% and 1–2% of Caucasians, respectively, and confer approximately 5-fold and 2-fold increases in the risk of incident venous thromboembolism (VTE). While some families who carry these genetic thrombophilia traits exhibit a prothrombotic phenotype, others have no (or only a limited) history of VTE. The ability to discern which individuals with personal and familial carriage of these genetic thrombophilias possess a clinically meaningful increase in VTE risk remains elusive, and (particularly among children) is perhaps best informed presently by family history of VTE.OBJECTIVE AND HYPOTHESES: We sought to evaluate overall plasma coagulative capacity in FV Leiden and PT G20210A heterozygotes using the Clot Formation and Lysis (CloFAL) assay, a global turbidimetric plasma assay of tissue-factor induced fibrin clot formation and tissue-type plasminogen activator enhanced fibrinolysis. We hypothesized that children heterozygous for either thrombophilia would not uniformly demonstrate hypercoagulability, but that coagulative capacity would be increased among heterozygotes who have a family history of VTE.PATIENTS AND METHODS: Children aged birth to 18 years (inclusive) enrolled in prospective inceptional cohort study of thrombosis/thrombophilia/stroke were included in the analysis if they were found to be heterozygous for FV Leiden or PT G20210A upon comprehensive thrombophilia testing and had undergone CloFAL assay testing on a research basis. Data on personal and family history of thrombotic events, thrombophilia testing, and CloFAL assay findings were analyzed. Intergroup comparisons of continuous data were performed by Mann-Whitney U test and proportions were compared between groups using chi-square or Fisher's exact test, as appropriate.RESULTS: Characteristics of the study population are shown in Table 1. Approximately 70% of patients were evaluated for a family history of VTE (with/without known thrombophilia) and nearly 50% had personal histories of VTE or arterial ischemic stroke (AIS)/recurrent transient ischemic attack (TIA); those evaluated for events were significantly older than those without events, and this difference was statistically significant among those with a positive family history fo VTE. Hypercoagulability was shown in 50% of patients and hypofibrinolysis in 13% using the CloFAL assay. Plasma coagulative capacity and maximal amplitude (MA) of the CloFAL waveform were significantly increased in patients with, versus without, family history of VTE (coagulation index, CI: 102% vs. 72% of the adult normal pooled plasma standard, respectively, p=0.04; MA: 0.415 vs. 0.322, p=0.02), and were not explained by age differences between groups. However, in this relatively small study population, the proportion of CloFAL CI results that exceeded the upper limit of normal values did not significantly differ between those with, versus without, family history of VTE. Pediatric FV Leiden or PT G20210A heterozygotes with positive family history of VTE were more likely to have multi-trait (>1) thrombophilia, in which case a trend toward increased plasma coagulability was demonstrated (CI: 139% [multi-trait] vs. 86% [isolated trait]; p=0.07); superimposed thrombophilias in this group most often consisted of elevated factor VIII activity and Lp(a) concentration.CONCLUSIONS: The present findings using the CloFAL global assay indicate that, while pediatric FV Leiden or PT G20210A heterozygotes do not uniformly exhibit hypercoagulability, plasma coagulative capacity is nevertheless significantly increased among heterozgyotes who have a family history of VTE, which may relate to the presence of superimposed thrombophilias.Table 1. Summary characteristics of the study population.N32Median age at evaluation (range)9.5 y (1–18 y)Personal history of events*14 y (1–18 y)No personal history of events*8 y (2–18 y)FV Leiden heterozygote (n)26**PT G20210A heterozygote (n)8**Personal history of VTE (n)11Personal history of AIS/recurrent TIA4Family history (1st/2nd degree) of VTE71%Multi-trait (>1) thrombophilia45%Acquired thrombophilia24%Hypercoagulability by CloFAL assay50%Hypofibrinolysis by CloFAL assay13%*VTE, AIS, or recurrent TIA**Two patients were dual heterozygotes.

Factor VLeiden and Prothrombin G20210A Gene Polymorphisms in Patients with Coronary Artery Disease

PurposeThe precise molecular mechanisms culminating in coronary artery disease (CAD) are not well understood, despite a wealth of knowledge on predisposing risk factors and pathomechanisms. CAD and myocardial infarction (MI) are complex genetic diseases; neither the environment alone, nor a single gene, cause disease, rather, a mix of environmental and genetic factors lead to atherosclerosis of the coronary arteries.Materials and MethodsIn the present study, our aim was to investigate the roles of prothrombin G20210A mutation and Factor VLeiden mutation in atherosclerotic coronary artery disease. 287 subjects (106 control subjects, who were angiographically normal, and 181 angiographically documented coronary atherosclerotic patients who exhibited coronary artery narrowing to a degree of ≥ 50%) were included in this study. The mutations were assessed with LightCycler Real-Time PCR mutation detection kits (Roche Diagnostics, GmbH, Germany).Results6.6% of control subjects, and 6.1% of patients with (50% coronary artery narrowing were determined to have the Factor VLeiden heterozygote mutation. 6.6% of control subjects had the Prothrombin G20210A heterozygote mutation, while 7.7% of patients with (50% coronary artery narrowing had this mutation. The OR for Factor VLeiden was 1.52 (CI: 0.240 - 9.602) and for Prothrombin G20210A mutation, the OR was 1.415 (CI: 0.287 - 6.962).ConclusionAlthough both the heterozygote Factor VLeiden and Prothrombin gene mutations were more frequent in patients with CAD than in control subjects, there was no statistical relationship found to exist between coronary artery disease and the Factor VLeiden and Prothrombin G20210A mutations.

Ocular Vascular Thrombotic Events: A Diagnostic Window to Familial Thrombophilia (Compound Factor V Leiden and Prothrombin Gene Heterozygosity) and Thrombosis

In a 12-member, 3-generation kindred with conjoint inheritance of G1691A factor V Leiden (FVL) and G20210A prothrombin gene (PTG) mutations, identified through a proband with amaurosis fugax and his father with nonarteritic ischemic optic neuropathy (NAION), the authors' hypothesis was that ocular thrombosis was a diagnostic window to familial thrombophilia-thrombosis. The authors used polymerase chain reaction (PCR) measures for thrombophilia (FVL, PTG, C677T-A1298C methylenetetrahydrofolate reductase [MTHFR], platelet glycoprotein PLA1A2) and hypofibrinolysis (plasminogen activator inhibitor-1 4G4G). The 39-year-old white male proband, with amaurosis fugax and transient ischemic attacks (TIA), was found to be a compound heterozygote for FVL and PTG mutations. His symptoms resolved only after coumadin. His 44-year-old brother (deep venous thrombosis [DVT]) and 46-year-old sister (DVT, pulmonary embolus [PE]) were compound FVL-PTG gene heterozygotes. Of 4 asymptomatic children born to these 3 siblings, 2 were FVL heterozygotes and 2 PTG heterozygotes. The proband's 69-year-old father, with NAION and ischemic stroke, had PTG heterozygosity, familial high factor VIII, and compound MTHFR C677T-A1298C mutation with homocysteinemia. The proband's 61-year-old aunt had PTG heterozygosity, recurrent DVT, and mesenteric artery thrombosis. The proband's 67-year-old mother, free of thrombotic events, was a FVL heterozygote, had high factor VIII, and PAI-1 4G4G homozygosity. In this extended kindred, ocular thrombotic events (amaurosis fugax, NAION) were associated with variegated thrombotic events, including TIA, ischemic stroke, DVT, PE, and mesenteric artery thrombosis, and opened a diagnostic window to family screening and treatment for complex thrombophilias, which had previously been undiagnosed.

Clinical evaluation of a functional prothrombin time-based assay for identification of factor V Leiden carriers in a group of Italian patients with venous thrombosis

The efficiency of a new prothrombin-based activated protein C (APC) resistance test to detect factor V Leiden (FVL) was clinically evaluated in 150 Italian patients with deep venous thrombosis. Patient samples are diluted in factor-V-deficient plasma, an APC-containing reagent, and specific factor V activator; after incubation, clotting is initiated by addition of activated-factor-FV-dependent prothrombin activator. Two prothrombin time determinations were performed under identical assay conditions except that no APC was added to one. A ratio over 4.2 for normal individuals and under 2.0 for FVL patients is expected: between 1.3 and 1.9 for FVL heterozygotes, and between 1.0 and 1.1 for FVL homozygotes. Using a predefined cut-off ratio of 2.0, a specificity and a sensitivity of 1.00 for detection of FVL mutation were found. With a cut-off ratio of 1.1, a specificity of 0.98 and a sensitivity of 1.00 were found for discrimination between FVL heterozygous (n = 60) and homozygous (n = 6). No interferences by heparins, oral contraceptives, oral anticoagulant therapy, protein C, protein S, D-dimer, homocysteine, MTHFR mutations and antiphospholipid autoantibodies were detected. In our experience, this new prothrombin time-based APC resistance assay provides improved discrimination between normal individuals and FVL carriers compared with the classical methods. Moreover, this new assay allows good discrimination between homozygous and heterozygous FVL carriers. In the authors' experience this prothrombin time-based method was not influenced by many factors compared with the classical activated partial thromboplastin time-based method.

Endogenous Thrombin Potential for Predicting Risk of Venous Thromboembolism in Carriers of Factor V Leiden

Measurement of endogenous thrombin potential (ETP) detects hypercoagulability and can be used to identify activated protein C resistance due to factor V Leiden (FVL). However, not all carriers of FVL suffer thrombosis and therefore we sought to determine if the test for ETP could be modified in such a way as to enable detection of FVL patients who were at increased risk of venous thromboembolism. Protac, an activator of both protein C and factor V, was incorporated into the traditional thrombin generation reaction and ratios (reaction with Protac:reaction without Protac) were calculated. Plasma samples from 42 FVL heterozygotes (12 with a history of thrombosis and 30 with no prior thrombosis) and 38 controls (non-FVL with no history of thrombosis) were analysed. The mean ETP ratio was significantly higher in FVL heterozygotes (0.90 ± 0.06) compared to normal controls (0.41 ± 0.10; p = 0.00004). Multivariate analysis indicated that the average ETP ratio was significantly and inversely correlated with factor V levels in FVL heterozygotes (p = 0.002) but not controls. Within the FVL group, patients with a history of thrombosis had higher ETP ratios (0.92 ± 0.06) compared to those without (0.89 ± 0.05), however, this did not reach statistical significance (p = 0.09). Further investigation into the use of ETP for detecting risk of thrombosis in people who are genetically predisposed is warranted. The recent introduction of diagnostic ETP measurements in the form of the calibrated automated thrombin generation from Thrombinoscope and the TechnoThrombin from Baxter should facilitate such studies.

Elevated factor VIII in hereditary haemorrhagic telangiectasia (HHT): Association with venous thromboembolism

Hereditary haemorrhagic telangiectasia (HHT) causes chronic nasal and gastrointestinal haemorrhage. Prothrombotic agents are commonly used for severe haemorrhage. Thrombotic risks have not been defined. In order to identify prothrombotic variables in HHT patients, and assess their potential functional significance, a pilot ELISA-based study comparing plasma proteins in healthy individuals with HHT to age/sex-matched non-HHT controls was validated in a full study of 309 consecutive HHT-affected individuals. In the pilot study, factor VIII (FVIII) and von Willebrand factor antigen concentrations were elevated in the HHT group compared to non-HHT controls (p<0.0013, Mann-Whitney). Service laboratory measurements confirmed high FVIII:Ag in 125 HHT-affected individuals with no recent ill-health, intervention or venous thromboemboli. FVIII:Ag levels increased with age. Logistic regression also suggested an age-independent association with HHT-associated pulmonary arteriovenous malformations (AVMs). No association was demonstrated between FVIII:Ag and acute phase response, disseminated intravascular coagulation, ABO group, pulmonary artery pressure, or markers of HHT haemorrhage. Elevated FVIII:Ag were associated with shortened activated partial thromboplastin times (APTTs), and VTE:VTE affected 20/309 (6.5%) HHT-affected individuals, at median age 61(36-71) years. Four VTE occurred in factorV Leiden heterozygotes in the months following PAVM-associated brain abscess. The strongest association with VTE was with log-transformed FVIII:Ag measured 10-132 months from VTE (odds ratio 2.41, 95% confidence intervals 1.254, 4.612, p=0.008). Age made no additional contribution to VTE risk once adjusted for FVIII:Ag. In conclusion, HHT-related elevation of FVIII:Ag levels may influence thrombotic risk in HHT. Individualised risk-benefit considerations may be helpful in HHT management.